During protein secretion in a fungal cell, certain proteins are cleaved by KEX2, a member of the KEX2 or “kexin” family of serine peptidase (EC 3.4.21.61). KEX2 is a highly specific calcium-dependent endopeptidase that cleaves the peptide bond that is immediately C-terminal to a pair of basic amino acids (i.e., the “KEX2 site”) in a protein substrate during secretion of that protein. KEX2 proteins generally contain a cysteine residue near the histidine residue of its active site and are inhibited by p-mercuribenzoate. The founding member of this group, the KEX2 peptidase of S. cerevisiae (Fuller et al., 1989, Proc. Natl. Acad. Sci. USA 86:1434-1438), cleaves the α-factor pheromone and killer toxin precursors during their secretion.
Production of fusion polypeptides has been reported in a number of organisms including E. coli, yeast and filamentous fungi. For example, bovine chymosin has been produced in Aspergillus niger as a fusion to full length glucoamylase (GAI) (Ward et al., (1990) Bio/technology 8:435-440; U.S. Pat. No. 6,265,204 and U.S. Pat. No. 6,590,078); human interleukin 6 (hIL6) has been produced in Aspergillus nidulans as a fusion to full-length A. niger glucoamylase (GAI) (Contreras et al., (1991) Biotechnology 9:378-381); hen egg white lysozyme (Jeenes et al., (1993) FEMS Microbiol. Lett. 107:267-273) and human lactoferrin (Ward et al., (1995) Bio/Technology 13:498-503) have been produced in Aspergillus niger as a fusion to residues 1-498 of glucoamylase; and bovine chymosin has been produced in Aspergillus niger as a fusion with full length native alpha amylase (Korman et al., (1990) Curr. Genet. 17: 203-212) and in Aspergillus oryzae as a fusion with truncated forms of A. oryzae glucoamylase (Tsuchiya et al., (1994) Biosci. Biotech. Biochem. 58: 895-899). Reference is also made to Shoemaker et al., 1981 Bio/Technology 1: 691-696; Nunberg et al., (1984) Mol. Cell. Biol. 4:2306-2315 and Boel et al., (1984) EMBO J. 3:1097-1102. In some of these fusion proteins, a KEX2 protease recognition site (Lys-Arg) has been inserted between a glucoamylase and a desired protein (e.g., Contreras et al., 1991 and Ward et al., 1995). The inventors of the present invention have found that protein secretion and/or protein cleavage may be enhanced in a fusion protein when the KEX2 recognition site has been manipulated to include an amino acid KEX2 site pre-sequence.
Specific literature of interest includes: Ward et al., (2004) Appl. Environ. Microbiol. 70:2567-2576; Goller et al., (1998) Appl. Environ. Microbiol. 64:3202-3208; La Grange et al., (1996) Appl. Environ. Microbiol. 62:1036-1044; Bergquist et al., (2002) Biochem. Biotechnol. 100:165-176; Spencer et al., (1998) Eur. J. Biochem. 258:107-112; Jalving et al., (2000) Appl. Environ. Microbiol. 66:363-368); Brenner and Fuller (1992) Proc. Natl. Acad. Sci. 89:922-926; Durand et al., (1999) Appl. Microbiol. Biotechnol. 52: 208-214; Ahn et al., (2004) Appl. Microbiol. Biotechnol. 64:833-839; Gouka et al., (1997) Appl Microbiol Biotechnol. 47:1-11 Broekhuijsen et al., (1993) J. Biotechnol. 31:135-145; MacKenzie et al., (1998) J. Biotechnol. 63:137-146 and published patent applications 20040018573 and 20050158825. Also U.S. Pat. No. 4,816,567 and U.S. Pat. No. 6,331,415 disclose processes for producing immunoglobulin molecules in recombinant host cells. The above cited literature is incorporated by reference herein for all purposes.
While numerous methods are available for the production of industrial enzymes and therapeutic proteins, there remains a need for alternative methods of protein production and particularly for therapeutic protein production, such as antibody production, which will result in relatively quick scale up time and high levels of produced protein with limited risk of contamination by viral or other adventitious agents. The present invention answers this need.